In the interdisciplinary field of tissue engineering, powerful new therapies are being developed to address structural and functional disorders of human health by utilizing living cells as engineering materials. See Viola et al., “The Emergence of Tissue Engineering as a Research Field,” prepared for the National Science Foundation, Oct. 14, 2003, available at nsf.gov. In some areas of tissue engineering, researchers are creating two- and three-dimensional tissues and organs from combinations of cells in order to repair or replace diseased or damaged tissues.
In instances where normal tissue cannot be engineered with the available cells, or enhanced cellular function is required, alternative approaches such as growth factor supplementation, macromolecule treatment or gene modification may be necessary to achieve the desired functionality. Moreover, in tissue engineering, the transfection or delivery of genes, proteins, molecules, nanoparticles, drugs, etc. is becoming vital in order to facilitate the formation of functional tissues and organs.
Gene transfection techniques have been used in various areas of research to improve cell and tissue function. Although there are established methods in the art for delivering genes into cells, the application of existing techniques to tissue engineering is not ideal. An important goal in gene transfection is to achieve efficient gene delivery to a target cell population while preserving cell viability. Currently, the most widely used methods for gene transfection are viral transfection, microinjection, electroporation, and the gene gun.
Transfection using viral vectors is a technique in which nucleic acids to be delivered are inserted into a virus. The nucleic acids are transported into the nucleus after the viral carriers enter the targeted cells by docking mechanisms. Although viral transfection has a high efficiency rate, it also has many drawbacks, such as residual pathogenicity, host immune response, and the potential induction of neoplastic growth following insertional mutagenesis. These concerns have restricted its application in medicine and in the biomedical areas, especially in clinical gene therapy.
Microinjection is another available technique. Genetic materials can be injected directly into cultured cells or nuclei by using microinjection needles. It is very effective in transferring specific genetic materials into the cells, and has been widely used in stem cell nuclear transfer applications. However, it is not efficient and not appropriate for studies and applications that require a significant number of cells to become transfected.
Electroporation, the application of controlled electric fields to facilitate cell permeabilization, is also used to enhance gene uptake into cells. The mechanism for entry is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of the DNA. This technique requires optimization for the duration and strength of the pulse for each type of cell used, and requires a critical balance between allowing efficient delivery and killing cells. Low cell viability is a major limitation of transfection by electroporation.
Finally, the gene gun can achieve direct gene delivery into tissues or cells by shooting gold particles coated with DNA at the cells. This technique allows direct penetration through the cell membrane into the cytoplasm and even the nucleus, bypassing the endosomal compartment of the cell. However, this method is limited by a low transfection efficiency.
Therefore, there is a need for new methods to effectively and efficiently transfect cells with nucleic acids, proteins, molecules, nanoparticles, drugs, etc. while protecting their viability. There is also a need to combine transfection with cell delivery, and further to combine these techniques into one platform or device for efficient and effective transfection in tissue engineering applications.